Recent work analyzing the eukaryotic transcriptome revealed the surprising observation of widespread, non- coding RNA (ncRNA) expression. Found globally throughout the genome, a few ncRNAs, such as Xist and HOTAIR, have been found to be involved in key developmental processes and disease states, such as X- chromosome inactivation and cancer, respectively. Consistent with this notion, ncRNAs have been found to be capable of dramatically altering gene expression patterns, sometimes by the recruitment of chromatin modifying enzymes. ncRNAs have also been found to be heavily-conserved throughout eukaryotes. While, collectively, this all suggests that ncRNAs play significant biological roles, the functions of the vast majority of ncRNAs are still unknown. One such report identified ~1,000 ncRNAs in the budding yeast, Saccharomyces cerevisiae, originating cryptically, both intergenically (between genes) and intragenically (within genes), and in the sense and anti-sense orientations, the majority of which have entirely unknown functions. Furthermore, even less is known of ncRNA regulation, despite the well-established roles of select ncRNAs. More recently, misregulation of ncRNAs has been associated with increased incidence of tumor metastasis in mice. This proposal, entitled Regulatory Mechanisms and Biological Functions of non-coding RNA, seeks to elucidate the molecular underpinnings behind the regulation of ncRNAs, as well as any biological functions that might underlie their widespread expression. Given their relevance to tumor metastasis, understanding their biological function and regulation cannot be understated. The proposal mainly employs molecular genetic techniques to: 1) uncover the mechanism by which ncRNAs negatively regulates mRNA expression and 2) identify genome-wide suppressors of ncRNA transcription, in the budding yeast, Saccharomyces cerevisiae. Towards the first aim, using microarray analysis, we already have candidate genes throughout the genome whose mRNAs seems to be negatively regulated by the overlapping ncRNA, termed transcriptional interference. Subsequent Gene Ontology (GO) analysis uncovered several pathways involved in growth regulation where this kind of regulatory mechanism seems to be commonly used. We will systematically rank these genes, and employ a series of genetic constructs to uncover the mechanism by which these genes become negatively regulated. In the latter aim, we have performed systematic genetic analysis to identify, in a high-throughput and unbiased manner, 308 putative ncRNA repressors. S. cerevisiae's genetic tractability make it especially amenable to a screen like this, which has not been previously developed. Ultimately, we would like the work outlined in this proposal to shed light on how ncRNAs are capable of promoting distinct gene expression patterns, which can reveal the molecular basis underlying human disease states.